Identification of toxin-binding protein involved in resistance to Cry1 toxins, and related screening methods

ABSTRACT

The subject invention relates in part to the surprising and unexpected discovery that insects that are resistant to  Bacillus thuringiensis  Cry toxins have measurably altered alkaline phosphatase (ALP) activity as compared to insects that are susceptible to Cry toxins. This and other surprising discoveries reported herein have broad implications in areas such as managing and monitoring the development of insect resistance to B.t. toxins. For example, the subject invention provides a simple and fast assay (enzymatic or otherwise) for detecting ALP activity levels and thereby monitoring the development of resistance by insects to crystal protein insect toxins. There was no prior motivation or suggestion to go about resistance monitoring using this simple and easy approach.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application Ser. No.60/490,119, filed Jul. 25, 2003.

BACKGROUND OF THE INVENTION

Billions of dollars are spent each year in efforts to control insectsthat damage crops and threaten food supplies. One alternative to the useof synthetic chemical pesticides are naturally occurring insecticidalcrystal protein (Cry) toxins from the bacterium Bacillus thuringiensis(B.t.). In order to preserve Cry proteins as a viable option for pestcontrol in years to come, efforts are being made to prevent theirover-use, as the development of resistance to Cry proteins by someinsect strains has been observed under certain conditions. The two maininsects that are currently known to develop resistance to Cry proteinsare the diamondback moth (DBM; Plutella xylostella) and the tobaccobudworm (Heliothis virescens).

In understanding how these and other insects might develop resistance toCry proteins, the mechanism(s) of action of Cry proteins is beinginvestigated. Specific binding to insect midgut receptors is a key stepin the mode of action of Cry proteins. Despite exceptions [1], in mostcases Cry toxin specificity and potency correlate with the extent oftoxin binding to midgut brush border membrane receptors in vitro [2, 3].Effective toxin binding to receptors results in toxin insertion andoligomerization on the midgut cell membrane, leading to pore formationand cell death by osmotic shock [4].

In brush border membrane vesicles (BBMV) from Heliothis virescenslarvae, three groups of binding sites (A, B, and C) for Cry1A toxinswere proposed based on their toxin binding specificities [5, 6]. The Abinding sites, which bind Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa and Cry1Jatoxins, include the cadherin-like protein HevCaLP (Jurat-Fuentes et al.,in preparation) and a 170-kDa N-aminopeptidase (APN) [5, 7-9].Currently, there is evidence that both HevCaLP [10] and the 170-kDa APN[8, 10] function as Cry1A toxin receptors, and knockout of HevCaLP, aprotein predicted to function in cell adhesion processes, results inCry1 resistance in larvae of YHD2 strains of H. virescens [10]. In the Bbinding site group, a 130-kDa protein has been shown to recognize bothCry1Ab and Cry1Ac. The C binding site group includes Cry1Actoxin-binding proteins smaller than 100-kDa in size [5].

Cry1 toxin-binding proteins of 60- to 80-kDa in size have been describedin toxin overlays of BBMV proteins from H. virescens [5], Manduca sexta[1], and Plodia interpunctella [12]. In 2D proteomic analysis of M.sexta BBMV proteins, McNall and Adang [13] reported Cry1Ac binding to aform of alkaline phosphatase (ALP, EC 3.1.3.1). Membrane bound ALP fromBombyx mori and M. sexta are attached to the brush border cell membraneby a glycosylphosphatidylinositol (GPI) anchor [13-15]. Specificinteractions between Cry1Ac and ALPs under native conditions resultinghave been reported for M. sexta [16] and H. virescens [17].

Altered glycosylation of 63- and 68-kDa glycoproteins was proposed asthe reason for reduced binding of soybean agglutinin (SBA) in H.virescens YHD2 strain, which are resistant to Cry1Ac [11]. However, acorrelation between a reduction in the amount of the 68 kDa protein andthe development, by insects, of resistance to B.t. Cry proteins wasnever before suggested or investigated. Furthermore, a link betweenmembrane-bound alkaline phosphatases (and associated levels of enzymeactivity) and the development of resistance by insects to Cry proteinshas never been proposed or suggested.

BRIEF SUMMARY OF THE INVENTION

The subject invention relates in part to the surprising and unexpecteddiscovery that insects that are resistant to Bacillus thuringiensis Crytoxins have measurably altered alkaline phosphatase (ALP) activity ascompared to insects that are susceptible to Cry toxins. This and othersurprising discoveries reported herein have broad implications in areassuch as managing and monitoring the development of insect resistance toB.t. toxins. For example, the subject invention provides a simple andfast assay (enzymatic or otherwise) for detecting ALP activity levelsand thereby monitoring the development of resistance by insects tocrystal protein insect toxins. There was no prior motivation orsuggestion to go about resistance monitoring using this simple and easyapproach.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A-E) illustrates identification of the 68-kDa BBMV glycoproteinas HvALP, a form of alkaline phosphatase.

FIG. 2 illustrates analysis of oligosaccharides on HvALP by lectinblotting.

FIG. 3(A-B) shows investigation of Cry1Ac binding to N-linkedoligosaccharides on HvALP.

FIG. 4(A-C) illustrates a comparison of HvALP levels and alkalinephosphatase activity between BBMV from susceptible and resistant H.virescens larvae.

FIG. 5 shows Cry1Ac ligand blots (A and B), HvALP immunodetection (C),and identification of HvALP protein spots from a 2D gel (D) by PMFsearches. (E) shows detection of HvALP by soybean agglutinin (SBA) orsera against mALP (HvALP) in BBMV from different H. virescens strains.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention generally relates, in part, to assays formonitoring the development by insects of resistance to crystal proteininsect toxins (Cry proteins), such as Cry proteins from Bacillusthuringiensis (B.t.). These assays can be practiced in the form ofsimple kits that are preferably used in the field to screen for thepresence of resistant insects. In preferred embodiments, levels ofenzyme activity or amounts of enzyme from gut cell membranes areassessed, relative to the levels and amounts from known susceptibleinsects, for determining the presence or absence of resistant insects.In further preferred embodiments, the enzyme is an alkaline phospatase.In still further preferred embodiments, the alkaline phosphatase is areceptor that binds a Cry protein.

To provide more context for this invention, it should be noted that itwas proposed that changes in glycosylation of 63- and 68-kDa midgutglycoproteins in the tobacco budworm (Heliothis virescens) correlatedwith reduced binding (of SBA) and the development of resistance, by H.virescens, to the Cry1Ac toxin from (B.t.). [11]. The subject inventionstems in part from the unexpected and surprising findings that this68-kDa protein from H. virescens is a membrane-bound, GPI-anchored formof alkaline phosphatase (ALP). This protein is further identified as areceptor for Cry1Ac. This receptor protein is thus referred to herein asHvALP (for H. virescens alkaline phosphatase). HvALP is identifiedherein as being involved in Cry1Ac toxicity to H. virescens larvae.Still further surprising is the discovery, as reported herein, thatinsect resistance to B.t. toxins correlates to altered levels ofactivity and/or amounts of membrane-bound ALP. This finding has broadimplications for areas such as managing insect resistance to B.t.toxins. For example, the subject invention provides a simple and fastassay (enzymatic or otherwise) for detecting and monitoring thedevelopment of resistance by insects to crystal protein insect toxins.There was no prior motivation or suggestion to go about resistancemonitoring using this simple and easy approach.

This invention stems in part from the observation that changes inalkaline phosphatase contribute to insects developing resistance to Cryproteins. It was proposed that a specific isoform of alkalinephosphatase binds Cry1Ac, and this receptor is modified in YHD2resistant larvae (a B.t.-resistant strain of H. virescens). The subjectinvention relates in part from the discovery that HvALP activity islower in brush border membrane vesicles from the YHD2 strain.

Ligand and lectin blots together with glycosidase digestion assaysrevealed that the existence of N-linked oligosaccharides containingterminal N-Acetylgalactosamine (GalNAc) residues on HvALP was necessaryfor Cry1Ac binding. Results reported herein indicate that reducedsoybean agglutinin (SBA) binding to HvALP from Cry1Ac resistant larvaewas surprisingly attributable to reduced amounts of HvALP in resistantlarvae. Immunoblotting and specific alkaline phosphatase activity ofBBMV proteins from susceptible and resistant larvae indicated thatdecreased HvALP levels were produced in YHD2 larvae. Quantification ofspecific alkaline phosphatase activity in brush border membrane proteinsfrom susceptible (YDK and F1 generation from backcrosses) and resistantYHD2 H. virescens larvae confirmed the reduced HvALP levels observation.

Accordingly, the subject invention provides methods for assaying anddetecting altered ALP levels (including detecting less ALP activity) ina suitable sample (a membrane preparation from a lepidopteran pest, suchas virescens, in preferred embodiments), as compared to ALP levels innon-resistant insects. The presence of an unexpected level of ALPindicates a B.t.-resistant insect. The subject invention provides asimple and fast assay (enzymatic or otherwise) for detecting andmonitoring the development of resistance by insects to crystal proteininsect toxins. There was no prior motivation or suggestion to go aboutresistance monitoring using this simple and easy approach.

Prior to the subject invention, it was never suspected or suggested thatthe reduced binding was due to an alteration to or a reduction in thelevels (amount or activity) of the subject ALP receptors. As explainedin more detail below, receptors generally serve important cellfunctions. Thus, one would not have expected, and it was verysurprising, to find resistance to be associated with absence orreduction of this receptor. There was no prior motivation to screen, inthe context of tracking B.t. resistance, an insect for altered orreduced levels of ALP receptors as discussed in detail herein.

It is interesting to note that in whole-insect brush border membranevesicle (BBMV) preparations obtained from Plutella xylostella (thediamondback moth or DBM), alkaline phosphatase activity was found to behigher in both the homogenate and BBMV of resistant insects compared tosusceptible insect strains. In-gel activity assays of SDS-PAGE separatedBBMV shows higher activities for both whole-insect and gut BBMVpreparations of resistant insects compared to the correspondingsusceptible preparations. Without being bound by a specific theoryregarding mechanism of action, it is possible that resistant DBM havetwo forms of ALP, and the B.t. receptor form is “shed” and anothernon-binding form is present in greater amounts. With this in mind, it isnow possible, according to the subject invention, to design antibodies(or other probes), for use in assays discussed herein, that bindspecifically (only) to the Cry binding form of ALP (in a particularinsect) and not to other forms of ALP.

The initial hypothesis to explain reduced Cry1Ac and SBA binding in YHD2larvae was based on possible alteration of protein glycosylation inresistant insects [11]. Surprisingly however, presently disclosedresults from immunoblotting and alkaline phosphatase activity detectionrevealed instead that HvALP protein levels were decreased in BBMV fromYHD2 larvae. Therefore, decreased SBA binding to HvALP from YHD2vesicles was presently, and surprisingly, found to be due to reducedprotein levels rather than altered glycosylation. Although due tolimiting YHD2 materials, oligosaccharide analysis was only performed inBBMV from YDK larvae, hence potential alterations of HvALP glycosylationin YHD2 larvae cannot be completely ruled out. In any case, consideringthat F1 generation larvae bound Cry1Ac toxin and were only two foldresistant to Cry1Ac [11], the present results show a direct correlationbetween decreased HvALP levels and increased resistance to Cry1Ac. (BBMVfrom the F1 generation of reciprocal crosses recovered HvALP levelsobserved for the susceptible parents independently of the sex of thesusceptible progenitor, demonstrating autosomal recessive transmissionof this trait.) While YHD2 larvae might have multiple resistancemechanisms, what is important is the subject discovery of the linkbetween reduced levels of this 68 kDa protein and resistance, which wasnever heretofore suggested.

Electrophoretic variations of alkaline phosphatase between differentstrains or developmental stages have been reported for Drosophilamelanogaster [54], Aedes aegypti [55], and B. mori [56, 57], althoughthe physiological consequences of these variations are not clearlyunderstood. In the Tsunomata B. mori strain, reduced mALP activitycorrelated with undetectable levels of mALP antigen, while there were noalterations in gene copy or transcript size [57]. These resultssuggested that electrophoretic mALP polymorphisms were due topost-transcriptional processes.

Insect alkaline phosphatases have been proposed to function in activeabsorption of metabolites and transport processes [29], although thereis also evidence for participation in cell adhesion and differentiation[59]. According to these important functions, significant fitness costsassociated with reduced ALP activity would be expected. Thus, one wouldnot have expected viable insects to develop resistance by eliminating orreducing membrane-associated ALP.

The specific mechanism by which YHD2 larvae reduce HvALP expression canbe further investigated. As stated above, if information from B. morimALP could be applied to HvALP, the decreased activity observed invirescens might not be related to changes in gene copy number ortranscription. An alternative hypothetical mechanism to reduce receptorsin midgut brush border membranes was previously proposed by Lu and Adang[60]. According to this hypothesis, GPI-anchored proteins would beselectively solubilized by endogenous PIPLC digestion in Bt-resistantinsects. Such treatment would result in elimination of potential Crytoxin binding sites, such as aminopeptidases, from the midgutepithelium. In support of this hypothesis, B. mori mALP is solubilizedby midgut epithelium enzymes to form digestive fluid alkalinephosphatase (dALP), which is highly resistant to degradation by midgutproteases [61].

In any case, the subject results demonstrate a direct correlationbetween decreased HvALP levels and Cry resistance in H. virescens. HvALPmay be a critical component in toxicity, or alternatively, the reducedHvALP levels observed in resistant larvae may indicate broaderalterations in the brush border membrane. One possibility is thatresistant larvae have altered membrane components such as lipid raftsthat affect the amounts of HvALP localized to the brush border membrane.The specific role of HvALP in Cry1Ac intoxication can be investigatedfurther.

Whatever the exact mechanisms of action are, HvALP is clearly identifiedherein as a resistance marker, so biochemical and DNA-based tests maynow be developed to detect emergence of resistance to B.t. crops infield populations. Sample insects can be collected in many ways frommany different locations. The subject invention relates to the discoverythat ALP is a membrane receptor for Cry toxins, and insects such asHeliothis virescens can evolve resistance to Cry1Ac, for example, bysomehow shedding this receptor (i.e., by effectively reducing the amountof this enzyme in their gut/on gut cells). These surprising discoverieshave broad implications in areas such as managing insect resistance toB.t. toxins. For example, insects can be screened for the presence orabsence of resistance by, for example, isolating membrane proteins andscreening them for the presence or absence of the activity associatedwith ALP. The presence or absence of the activity of this protein canalso be screened directly (without first isolating membrane proteins).

As will be recognized in the art in light of the subject disclosure, thesubject invention is not limited to screening Heliothis virescens. Otherinsects, including Plutella xylostella (diamondback moths) and Manducasexta, can also be screened with methods and apparatuses of the subjectinvention. Lepidopterans are preferred screening targets, but otherinsects can also be screened according to the subject invention. Inlight of the subject discovery, it will now be known that decreasedamounts of ALP receptors can be an indicator of insects developingresistance to crystal protein insect toxins.

Furthermore, the subject invention is not limited to Cry1Ac receptorsand managing and/or monitoring resistance to Cry1Ac. Resistance to otherinsect toxin proteins can also be assessed according to the subjectinvention. Cry1A toxins (such as 1Aa, 1Ab, and 1Ac) are one preferredgroup of toxins for which the development of resistance thereto can bemonitored according to the subject invention. Other Cry proteins areidentified in “Revision of the Nomenclature for the Bacillusthuringiensis Pesticidal Crystal Proteins,” N. Crickmore, D. R. Zeigler,J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H.Dean, Microbiology and Molecular Biology Reviews (1998) Vol 62:807-813.It is also available online.

Detecting decreased (or altered) levels of ALP in an insect's gut, gutmembrane, and/or gut cell membranes can be done in a variety of ways.One way is by detecting ALP enzymatic activity in a suitable activity.Antibodies (monoclonal or polyclonal) to ALP can also be used indetection methods. Methods such as ELISA are well-known in the art.Antibodies can also be linked to another type of detectable label, suchas a fluorescent label. Thus, the level of bound fluorescent antibodycan be assessed. Levels of RNA, for example, can also be detected. Asalkaline phosphatase is a known enzyme, and the sequences of some genesthat encode ALP are available in GENBANK, suitable nucleic acid probescan be designed for use in detection (hybridization) methods of thesubject invention. For example, sequence corresponding to 260 aminoacids of Bombyx mori alkaline phosphatase has been cloned. Thus, forsome aspects of this invention (e.g., for some screening methods),various known forms of this protein can be used.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Insect Strains and Brush Border Membrane Vesicle (BBMV)Preparation

H. virescens laboratory strains YDK and YHD2 have been previouslydescribed [18]. YDK is the unselected susceptible control colony for theCry1Ac-selected YHD2 strain, which developed 10,000-fold resistance toCry1Ac when compared to susceptible YDK larvae [19]. After continuousselection with Cry1Ac, levels of resistance increased to 73,000-fold[11]. Fifth instar larvae from each strain were dissected and midgutsfrozen and kept at −80° C. until used to prepare BBMV.

BBMV were isolated by the differential centrifugation method ofWolfersberger et al. [20]. BBMV proteins were quantified by the methodof Bradford [21], using BSA as standard, and kept at −80° C. until used.N-aminopeptidase (APN) activity using leucine-p-nitroanilide as thesubstrate was used as a marker for brush border enzyme enrichment in theBBMV preparations. APN activities were enriched 6-8 fold in the BBMVpreparations compared to initial midgut homogenates.

Example 2 Cry1Ac Toxin Purification and Labeling

B. thuringiensis strain HD-73 obtained from the Bacillus Genetic StockCenter (Ohio, USA) was used to produce Cry1Ac. MutatedCry1Ac⁵⁰⁹QNR⁵¹¹-⁵⁰⁹AAA⁵¹¹ was expressed in Escherichia coli MV 1190kindly provided by Dr. Donald Dean (Ohio State University, Ohio, USA),and purified as described elsewhere [22]. This Cry1Ac mutant toxin lacksthe GalNAc binding properties of the wild type toxin [23]. Cry1Accrystalline inclusions were solubilized, activated and purified aspreviously described [24]. Purified toxin samples (verified by reducingSDS-10% PAGE) were pooled, protein concentration determined as for BBMVproteins and stored at −80° C. until used.

Purified Cry1Ac (1 μg) was radiolabeled with 0.5 mCi of Na¹²⁵I by thechloramine T method [1]. Specific activities of labeled samples were 3-8mCi/mg, as determined using the bindability method of Schumacher et al.[25]. Labeled toxins were kept at 4° C. and used within 10 days.

Example 3 Identification of the 68-kDa BBMV Glycoprotein as AlkalinePhosphatase

FIG. 1 illustrates identification of the 68-kDa BBMV glycoprotein asHvALP, a form of alkaline phosphatase. BBMV proteins from H. virescensstrains specified in FIG. 1 were separated by electrophoresis andCoomassie blue stained to control for equal protein loads (FIG. 1A) ortransferred to PVDF filters. After blocking, filters were probed withSBA lectin (FIG. 1B) or sera against the membrane bound form of ALP(mALP) from B. mori [27]. See FIG. 1C. Blots were developed usingenhanced chemiluminescence. Alkaline phosphatase activity in separatedBBMV proteins (FIG. 1D) was detected by incubating filters in NBT-BCIPuntil purple precipitate was visualized in the region of enzymaticactivity. For detection of GPI-anchored proteins in BBMV protein blots(FIG. 1E), protein blots were treated with PIPLC and cleaved GPI anchorsdetected by probing with sera against the CRD determinant. BBMV proteinscontaining cleaved GPI anchors were visualized by enhancedchemiluminescence. The arrow indicates electrophoretic position of HvALPon the filters.

Although no protein amount differences were detected in Coomassie bluestained gels (FIG. 1A), the 68-kDa protein had reduced soybeanagglutinin (SBA) binding in BBMV from YHD2 larvae (FIG. 1B). Thisprotein was recognized by sera against mALP (FIG. 1C) and displayed ALPactivity in blots of BBMV proteins (FIG. 1D), demonstrating that thisprotein is a form of alkaline phosphatase. PIPLC digestion was used todetermine whether the 68-kDa protein was GPI anchored to BBMV in H.virescens. As shown in FIG. 1E, after PIPLC digestion, anti-CRD serarecognized the 68-kDa protein in H. virescens BBMV, suggesting that thisprotein is GPI-anchored to the brush border membrane. These results alsoshow that the 68-kDa protein with altered glycosylation in theCry1Ac-resistant YHD2 larvae was a form of ALP. Based on these results,the 68-kDa GPI-anchored glycoprotein was named HvALP for H. virescensalkaline phosphatase.

Example 4 Quantification of Alkaline Phosphatase and AminopeptidaseActivities

Specific alkaline phosphatase (ALP) and aminopeptidase-N (APN) enzymaticactivities of BBMV proteins were measured using p-nitrophenyl phosphatedisodium (pNPP) and leucine-p-nitroanilide (Sigma, St. Louis, Mo., USA)as substrates, respectively. BBMV proteins (1 μg) were mixed with ALPbuffer (100 mM Tris/HCl pH 9.5, 100 mM NaCl, 5 mM MgCl₂) or PBS buffer(10 mM Na₂HPO₄ pH 7.5, 135 mM NaCl, 2 mM KCl) containing 1.25 mM pNPP or0.8 mM leucine-p-nitroanilide, respectively. Enzymatic activities weremonitored as changes in OD at 405 nm wavelength for 5 minutes at roomtemperature (ALP) or at 37° C. (APN) in a microplate reader (MolecularDevices). One enzymatic unit was defined as the amount of enzyme thatwould hydrolyze 1.0 μmole of substrate to chromogenic product per minuteat the specific reaction pH and temperature. Data shown are the meanspecific activities from at least four independent BBMV batches fromeach H. virescens strain measured in at least three independentexperiments.

Example 5 Importance of ALP Glycosylation for Cry1Ac Binding; Ligand,Lectin and Immunoblots of BBMV Proteins

To investigate the oligosaccharides present on HvALP from Cry1Acsusceptible larvae, lectin blotting was performed using selected lectins(Table 1) and BBMV proteins from YDK larvae. After lectin blotting,HvALP on blots was detected by sera against B. mori mALP to confirmlectin binding to HvALP.

BBMV proteins (15 or 2 μg) were separated by SDS-PAGE 8%, and gels wereeither stained or electrotransferred to polyvinylidiene difluoride Q(PVDF) membrane filters (Millipore). After overnight transfer, filterswere blocked for one hour at room temperature with PBS buffer containing0.1% Tween-20 (PBST) and 3% BSA.

For immunoblots, blocked filters were probed with a 1:25,000 dilution ofpolyclonal serum against the membrane bound form of alkaline phosphatase(mALP) from B. mori (kindly provided by Dr. Masanobu Itoh, KyotoInstitute of Technology, Kyoto, Japan) for one hour. (It should be notedthat B. mori ALP is one of the few known insect ALPs available, and thatthese antibodies were not previously used in work in any way associatedwith studying B.t. proteins.) After washing with PBST containing 0.1%BSA, blots were probed with anti-rabbit serum (Sigma) conjugated tohorseradish peroxidase (HRP) or alkaline phosphatase. Filters weredeveloped using enhanced chemiluminescence (ECL, Amersham BioSciences)for peroxidase conjugates, or nitroblue tetrazolium (NBT) and5-bromo-4-chloro-3-indolyl-phosphate (BCIP) for alkaline phosphataseconjugates. No endogenous alkaline phosphatase activity was detectedwith NBT-BCIP in blots of BBMV proteins when samples were boiled beforeelectrophoresis. Periodate oxidation treatment of blots prior toimmunoblotting did not alter antigenicity of BBMV proteins, showing thatthe serum used recognized protein and not sugar epitopes.

For lectin blots, blocked filters containing separated BBMV proteinswere incubated with lectins from Canavalia ensiformis (ConA, at 0.05μg/ml), Artocarpus integrifolia (Jac, at 0.5 μg/ml), Glycine max (SBA,at 1 μg/ml), Ricinus communis (RCA-I, at 5 μg/ml), Dolichus biflorus(DBA, at 5 μg/ml), Sophora japonica (SJA, at 5 μg/ml), Wistariafloribunda (WFL, at 1 μg/ml), Helix pomatia (HPL, at 1 μg/ml), orGriffonia simplicifolia (GSL-I, at 5 μg/ml) for one hour in blockingbuffer (PBST plus 3% BSA). Con A, Jac, SBA, and HPL were purchased fromSigma; RCA-I, SJA, WFL, and GSL-I were from Vector laboratories(Burlingame, Calif., USA).

Lectins conjugated to HRP were visualized by enhanced chemiluminescence(ECL). Blots of biotinylated lectins were probed with streptavidin-HRPconjugate (Vector) and then visualized as HRP-conjugated lectins. Ascontrols for non-specific lectin binding, lectins were incubated withspecific hapten sugars (Table 1) for 30 min. at room temperature beforeprobing BBMV blots. This treatment eliminated or greatly decreasedlectin binding to BBMV proteins on filters. See FIG. 2, whichillustrates analysis of oligosaccharides on HvALP by lectin blotting.BBMV proteins from YDK larvae were separated by electrophoresis andtransferred to PVDF filters. After blocking, filters were probed withspecific lectins as indicated in the figure. Lane 1: bound lectins werevisualized by enhanced chemiluminescence. Lane 2: immunodetection ofHvALP using sera against the mALP from B. mori. HvALP was visualized byanti-rabbit-alkaline phosphatase conjugate and NBT-BCIP, so that bothlectin blots and HvALP immunodetection could be performed using the samefilter. Lane 3: competition of lectin binding with the respective haptensugar (See Table 1, below). For release of N-linked oligosaccharidesfrom BBMV proteins (PNG-F/SBA), filters were treated withpeptide-N-glycanase F (PNG-F). After washing, filters were probed withSBA and developed as for SBA lectin blots. All treatments werereplicated at least thrice to confirm reproducibility.

Table 1 shows sugar specificities of lectins (based on [62]) used inblots and respective hapten sugars used for lectin specificity controls.Several lectins were selected according to their specificity of bindingto galactose (Gal), N-Acetylgalactosamine (GalNAc), N-Acetylglucosamine(GlcNAc), mannose (Man) or glucose (Glc).

TABLE 1 Lectin (abbreviation) Sugar specificity Hapten sugar Canavalisensiformis α-Man 0.2 M αmethylman/ (ConA) α-Glc glc Artocarpusintegrifolia Galβ₁→3GalNAc 0.8 M Gal (Jac) Galβ₁→3,4GlcNAc Glycine max(SBA) α/βGalNAc 0.2 M GalNAc α/βGal Ricinus communis (RCA-I)Galβ₁→4GlcNAc 0.2 M Gal Galα₁→3Gal Dolichus biflorus (DBA)GalNAcα₁→3GalNAc 0.2 M GalNAc GalNAcα₁→3Gal Sophora japonica (SJA)Galβ₁→3GalNAc 0.2 M Gal Galβ₁→3,4GlcNAc Wistaria floribunda (WFL)α/βGalNAc 0.2 M GalNAc Helix pomatia (HPL) GalNAcα₁→3GalNAc 0.2 M GalNAcGalNAcα₁→3Gal Griffonia simplicifolia GalNAcα₁→3Gal 0.2 M Gal (GSL)Galα₁→3,6Gal/Glc

For SBA binding competition, filters were blocked as above, and then 12μg/ml of Cry1Ac or the Cry1Ac mutant protein ⁵⁰⁹QNR⁵¹¹-⁵⁰⁹AAA⁵¹¹ wereadded to the blocking buffer along with SBA lectin (1 μg/ml). Afterone-hour incubation and washing, filters were developed as described forlectin blots.

Ligand blots were done as previously described [5]. ¹²⁵I-Cry1Ac (1 nM)was used to probe blotted BBMV proteins in blocking buffer for one hourat room temperature. After washing, filters were exposed to photographicfilm at −80° C. for 24 hours.

To detect HvALP in the filters used for lectin or ligand blotting, afterdevelopment, filters were washed in PBST plus 0.1% BSA overnight.Blocking and HvALP immunodetection were performed as described above. Toavoid interference with lectin or toxin detection, bound mALP antiserawas detected by anti-rabbit sera conjugated to alkaline phosphatase.

As shown in FIG. 2, HvALP was recognized by lectins from Canavaliaensiformis (ConA), Glycine max (SBA), and Wistaria floribunda (WFL). Thedifferent pattern of BBMV proteins being recognized by both SBA and WFL(both bind terminal GalNAc) was probably due to the existence ofterminal GalNAc in linkages poorly recognized by one of the lectins.Conversely, no binding to HvALP was detected using lectins fromArtocarpus integrifolia (Jac), Ricinus communis (RCA), Dolichus biflorus(DBA), or Helix pomatia (HPL). Although proteins of similar size toHvALP were bound by Griffonia simplicifolia (GSL) and Sophora japonica(SJA) lectins, immunodetection of HvALP in these filters demonstratedthat the detected lectin binding proteins were not HvALP. To furthertest the existence of terminal GalNAc on N-linked oligosaccharides onHvALP, we performed digestion of blotted BBMV proteins withpeptide-N-glycosidase-F (PNG-F), which releases N-linkedoligosaccharides as N-glycosides from polypeptide chains. Digestion ofBBMV proteins with PNG-F eliminated binding of SBA to HvALP (FIG. 2),supporting the hypothesis that this protein has N-linkedoligosaccharides with terminal GalNAc residues. Binding of SBA to otherBBMV proteins was also decreased after PNG-F digestion, suggesting thepresence of GalNAc or galactose on N-linked oligosaccharides in theseproteins. Thus, according to one aspect of the subject invention, aninsect (or insects) can also be screened for the presence or absence (orreduced amounts) of glycosylation to determine if the insect isresistant to crystal protein toxins (which would be indicated bydecreased amounts of bound lectins (preferably SBA).

Example 6 Digestion of BBMV Proteins with Peptide-N-Glycosidase F

Release of N-linked oligosaccharides from BBMV proteins was achieved bydigestion of blotted BBMV proteins with peptide-N-glycosidase F (PNG-F).BBMV proteins (15 μg) were separated by SDS-8% PAGE and transferred toPVDF filters as above. Filters were incubated in 5 ml of PBS buffer (pH7.4) containing 0.1% SDS, 0.5% Triton-X-100 and 30 units of PNG-F(Boehringer-Mannheim) for 17 hours at 37° C. After treatment, filterswere blocked and probed as for SBA lectin blots or ¹²⁵I-Cry1Ac ligandblots. Controls, which had no PNG-F in the incubation buffer, showed nodifferences in lectin or toxin binding when compared to SBA and¹²⁵I-Cry1Ac blots.

Example 7 Detection of GPI Anchors

The presence of glycosylphosphatidylinositol (GPI) anchors in BBMVproteins was detected following the method described by Luo et al. [8].Briefly, after phosphatidylinositol-specific phospholipase C (PIPLC)digestion of BBMV blots, cleaved GPI anchors were detected byimmunological detection of the exposed cross-reacting determinant (CRD)epitope contained in the residue of the GPI anchor by probing withanti-CRD sera (kindly provided by Dr. Mensa-Wilmot, University ofGeorgia, Athens, Ga., USA). Blots were probed with anti-rabbit-HRPconjugate (Sigma) before developing with enhanced chemiluminescence asabove. In controls, which had no PIPLC in the blocking buffer, noproteins were detected.

Example 8 Detection of Alkaline Phosphatase Activity in SDS-PAGE Gelsand Blots

To detect alkaline phosphatase activity in BBMV, proteins (15 or 2 μg)solubilized in sample buffer [26] were not heat denatured before gelloading. After SDS-PAGE 8% electrophoresis and transfer to PVDF, filterswere washed with ALP buffer for 15 minutes at room temperature. Afteraddition of 330 μg/ml of NBT and 165 μg/ml of BCIP to the ALP buffer,alkaline phosphatase activity was visualized by the formation of apurple-red precipitate. Reactions were stopped by incubation of filtersin 50 ml of PBS pH 7.5 containing 200 μl of 500 mM EDTA pH 8.0.

Example 9 Importance of ALP Glycosylation for Cry1Ac Binding

To test the hypothesis that Cry1Ac toxin bound to the terminal GalNAcresidue on HvALP, SBA binding to HvALP was competed with Cry1Ac. Thereciprocal competition assay was not performed due to the 10⁶-fold loweraffinity of SBA for GalNAc (K_(d)=0.3 mM; [28]) when compared to Cry1Acaffinity for its binding sites (K_(d)=1.1 nM; [5]).

FIG. 3 shows investigation of Cry1Ac binding to N-linkedoligosaccharides on HvALP. For competition of SBA binding (A), blockedPVDF filters containing separated BBMV proteins from YDK larvae wereprobed with SBA lectin (SBA) or SBA lectin plus either Cry1Ac(Cry1Ac/SBA) or the Cry1Ac mutant ⁵⁰⁹QNR⁵¹¹-⁵⁰⁹AAA⁵¹¹ (QNR/SBA), whichlacks GalNAc binding. Bound SBA lectin was detected by enhancedchemiluminescence. For ligand blots (B), BBMV proteins binding Cry1Acwere detected by probing blocked filters with 1 nM ¹²⁵I-Cry1Ac for onehour (Cry1Ac). Importance of N-linked oligosaccharides for ¹²⁵I-Cry1Acbinding (PNG/Cry1Ac) was tested by digestion of BBMV proteins with PNG-Fglycosidase. After digestion, filters were washed, blocked and treatedas described for ligand blots. Bound toxin was detected byautoradiography. Asterisks indicate the electrophoretic position of the170- and 130-kDa proteins, arrows indicate the position of HvALP in thefilters. Radiography of the radiolabeled Cry1Ac toxin used for theseexperiments (¹²⁵I-Cry1Ac) is included.

When comparing SBA binding to BBMV (FIG. 3A) with Cry1Ac competitionblots (FIG. 3B), Cry1Ac prevented SBA binding to HvALP as well as toother BBMV proteins, indicative of toxin binding to terminal GalNAcresidues on these proteins. Binding of SBA to the 170-kDa APN was almostunaffected by the presence of Cry1Ac. As a control for toxin binding notdue to GalNAc recognition, we competed SBA binding with a Cry1Ac mutant,⁵⁰⁹QNR⁵¹¹-⁵⁰⁹AAA⁵¹¹, which lacks GalNAc binding [23]. SBA binding toHvALP was unchanged by ⁵⁰⁹QNR⁵¹¹-⁵⁰⁹AAA⁵¹¹ (FIG. 3C), demonstrating thatCry1Ac bound to terminal GalNAc on HvALP.

To provide further support for the hypothesis of Cry1Ac binding toGalNAc on HvALP, ligand blots were performed with ¹²⁵I-Cry1Ac. Cry1Acbound to several BBMV proteins, including HvALP (FIG. 3D). When N-linkedoligosaccharides were released from HvALP by PNG-F digestion, Cry1Ac didnot bind to this protein, demonstrating that toxin binding was dependenton the presence of N-linked oligosaccharides on HvALP. Binding to otherCry1Ac binding proteins was also greatly decreased by PNG-F digestion,indicating the importance of N-linked protein glycosylation for Cry1Acbinding on blots.

Example 10 Reduced HvALP Correlates with Resistance to Cry1Ac

To investigate the possibility that reduced SBA binding to HvALP fromYHD2 larvae (FIG. 1B) was a result of decreased HvALP protein levels,the following comparisons were conducted using immunodetection andalkaline phosphatase activity blots: HvALP from YHD2, YDK, and larvaefrom the F1 generation of backcrosses between YDK and YHD2 adults. Twodifferent types of F1 larvae, according to the sex of the susceptibleparent, were used to determine the potential existence of sex linkage.As shown in FIG. 4B, sera against the membrane-bound form of alkalinephosphatase from B. mori recognized HvALP in BBMV from YDK, YHD2 and F1larvae. No differences in intensity of recognition were observed betweenHvALP from YDK and F1 vesicles, while recognition of HvALP in YHD2 wasclearly reduced.

FIG. 4 illustrates a comparison of HvALP levels and alkaline phosphataseactivity between BBMV from susceptible and resistant H. virescenslarvae. BBMV proteins from YDK (lane 1), YHD2 (lane 2), F1 generation ofYDK males crossed with YHD2 females (lane 6), or F1 generation of YDKfemales crossed with YHD2 males (lane 7), were separated byelectrophoresis. For comparison, lanes 3, 4 and 5 contained YHD2 BBMVproteins at 3-, 5-, and 10-fold respectively the protein concentrationused for YDK and F1 lanes. Gels were Coomassie blue stained (panel A),or transferred to PVDF filters (panels B and C). After blocking, blot inpanel B was probed with sera against the mALP from B. mori to detectHvALP. For visualization of alkaline phosphatase activity (panel C), thefilter was washed in ALP buffer, and then NBT-BCIP included in thebuffer as described in Materials and Methods. Alkaline phosphataseactivity was visualized as a purple precipitate.

To confirm reduction in HvALP antigen in BBMV from YHD2, the proteinload was increased by 3-, 5- and 10-fold to compare to YDK and F1vesicles. Increased BBMV protein concentrations as observed in thestained gel (FIG. 4A) resulted in augmented HvALP recognition (lanes 3,4 and 5 in FIG. 4B), clearly suggesting a reduction in HvALP proteinlevels in BBMV from YHD2 larvae. Visual comparison of the lanes withincreasing YHD2 protein loads and the YDK and F1 lanes in the blots(FIG. 4B) suggested a 3- to 5-fold reduction in HvALP antigen levels inBBMV from YHD2 larvae when compared to YDK or F1 vesicle proteins.

Thus, it was predicted that reduced HvALP amounts in BBMV from YHD2larvae would result in reduced alkaline phosphatase activity. Alkalinephosphatase activity in blots of BBMV proteins from YDK and F1 larvaewas similar, and higher than activity in YHD2 vesicles (FIG. 4C). Inagreement with reduced protein levels observed in FIG. 4B, specificalkaline phosphatase activity in suspensions of BBMV from YHD2 insectswas reduced 3- to 4-fold when compared to YDK or F1 vesicles (see Table2, below). Aminopeptidase-N specific activity was used as control, withno significant differences found between BBMV from YDK, YHD2 or F1larvae.

Table 2 shows specific alkaline phosphatase (ALP) and aminopeptidase-N(APN) activities of BBMV suspensions from YDK, YHD2 and F1 larvae.Specific activity of BBMV suspensions is expressed in units permilligram of BBMV protein (U/mg). One enzymatic unit was defined as theamount of enzyme that would hydrolyze 1.0 μmole of substrate tochromogenic product per minute at the specific reaction pH andtemperature. SD=standard deviation of the mean based on at least sixindependent measurements.

TABLE 2 ALP activity APN activity BBMV sample (U/mg) ± SD (U/mg) ± SDYDK 223 ± 91 2192 ± 427 YHD2  77 ± 37 2364 ± 290 YDK♀ × YHD2♂ 375 ± 123156 ± 62  YHD2♀ × YDK♂ 292 ± 12 2921 ± 275

These results indicated that reduced amounts of HvALP in BBMV from YHD2larvae result in reduced alkaline phosphatase activity and correlatewith resistance to Cry1Ac and reduced Cry1Ac toxin binding.

Example 11 Further Characterization of the Nature of HvALP as a Cry1AcReceptor and Alteration of this Receptor in Resistant YHD2 Larvae

Two main approaches are discussed in this Example: (1) proteomicanalysis through 2D in-gel differential electrophoresis (2D-DIGE) ofsusceptible and resistant BBMV proteins to identify changes related toHvALP, and (2) studies on the molecular mechanism responsible forreduced HvALP levels in resistant larvae. Towards completion of (1),Peptide Mass Fingerprints (PMFs) of BBMV protein spots were identifiedas HvALP based on detection by sera against the mALP from Bombyx mori(FIG. 2C) and specific Cry1Ac binding on 2D ligand blots (FIGS. 2A and2B). Database searches using the obtained PMFs identified the proteinspots as membrane bound alkaline phosphatase. These results alsohighlight the utility of PMF database searches to identify proteinsseparated in 2D gels and identify HvALP as a Cry1Ac binding protein in2D ligand blots.

Relative to (2), in further studying HvALP alteration as a mechanism forCry1Ac resistance, HvALP in susceptible (YDK) and additional resistantH. virescens strains (CXC, and KCBhyb) was compared using SBA lectinblots, immunoblots with anti-mALP sera, and measurements of ALP activityin BBMV.

FIG. 5 shows Cry1Ac ligand blots (A and B), HvALP immunodetection (C),and identification of HvALP protein spots from a 2D gel (D) by PMFsearches. BBMV proteins from susceptible larvae were separated by 2Delectrophoresis, then stained with Sypro Ruby stain (D) or transferredto PVDF filters. After blocking, filters were probed with 5 nMbiotinylated Cry1Ac (A) and 500-fold excess unlabeled Cry1Ac ascompetitor (B) or with sera against the mALP from B. mori (C). Proteinspots from (D) were digested with trypsin and the resulting PMFs wereused for database searches. Identification of the spots is indicated.The Z value is a measure of probability (values higher than 2.3 denote95% confidence of correct match). (E) Detection of HvALP by soybeanagglutinin (SBA) or sera against mALP (HvALP) in BBMV from different H.virescens strains. Immunodetection of actin in the same samples was usedas loading control.

As shown in FIG. 5E, HvALP levels were reduced in BBMV from YHD2 andKCBhyb larvae when compared to YDK and CXC vesicles. SBA binding, whichreflects the presence of terminal GalNAc residues recognized by Cry1Ac(EJB manuscript), was greatly reduced in BBMV from all the resistantstrains.

Alkaline phosphatase (ALP) and aminopeptidase-N (APN) activities in BBMVfrom susceptible (YDK) and resistant (YHD2, CXC, KCBHyb) strains of H.virescens were assayed. In agreement with the HvALP alterations, ALPactivity was reduced in BBMV from YHD2 (129±65 U/mg), CXC (116±65 U/mg),and KCBhyb (123±67 U/mg) when compared to YDK (362±135 U/mg) vesicles.Assays were performed as described in Jurat-Fuentes and Adang (2004).Specific activity (±standard deviation) is expressed in units permilligram of BBMV protein (U/mg). One unit is defined as the amount ofenzyme that will hydrolyze 1.0 μmole of p-nitrophenyl phosphate (PNPP)to p-nitrophenol (PNP) and phosphate per minute at pH 9.5 at 25° C.

Therefore, these results suggest that alterations of (includingreductions in the amount of) HvALP (in protein amounts, activities,and/or glycosylation) correlate with resistance to Cry1Ac, and thoseHvALP alterations may be used as resistance markers.

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1. A method of screening a lepidopteran for resistance to a Bacillusthuringiensis (B.t.) insecticidal Cry1 protein that is toxic tolepidopterans, wherein said lepidopteran is of a species known todevelop resistance to said Cry1 protein, wherein said method comprisescollecting said lepidopteran from a field of a B.t. crop, obtaining agut cell membrane sample from said lepidopteran, adding a plurality ofdetectable anti-alkaline phosphatase antibodies to said sample whereinsaid antibodies bind to an approximately 68 kDa alkaline phosphataseenzyme, detecting a level of bound antibody, and comparing said level ofbound antibody to a control amount of bound said antibodies determinedfrom a gut cell membrane preparation from a control insect of knownsusceptibility to said Cry1 protein, wherein resistance to said Cry1protein by said lepidopteran is indicated if said level is less thanthat of a non-resistant insect, wherein said non-resistant insect is ofthe same genus and species as said lepidopteran.
 2. The method of claim1 wherein said lepidopteran is a Heliothis virescens.
 3. The method ofclaim 1 wherein said method comprises running said sample on a gel, andassessing the amount of an approximately 68 kDa protein band on said gelby the use of a plurality of anti-alkaline phosphatase antibodies. 4.The method of claim 1 wherein said lepidopteran is a Heliothis.
 5. Themethod of claim 1 wherein said lepidopteran is of the family Noctuidae.6. The method of claim 1 wherein said Cry1 protein binds an alkalinephosphatase insect receptor.
 7. The method of claim 1 wherein said Cry1protein is a Cry1Ac protein.